The main objective of the proposed research is to elucidate some of the mechanisms for the control and regulation of DNA-dependent RNA transcription in eucaryotic cells. The proposed experiments focus on the cellular components involved in the transcription of the chloroplast chromosome of Euglena gracilis. Molecular hybridizations will be employed to study changes in the expression of selected genes or groups of genes of the chloroplast genome during light-induced chloroplast development. Hybridization probes for specific DNA sequences, such as transfer RNAs, total chloroplast polysomal mRNA, and specific mRNAs (ribulose 1,5-diphosphate carboxylase large subunit mRNA) will be prepared by the techniques previously developed for chloroplast rRNA sequences. The probes are hybridized to excess cellular RNA from different developmental stages. From the kinetics of such reactions, a temporal program for transcription of a number of chloroplast genes during development will be determined. The aim is to correlate the in vivo transcription program with changes in the properties of a purified, transcriptionally active chromosome (TAC) isolated directly from Euglena chloroplasts. The TAC has previously been shown to selectively transcribe the chloroplast genome in vitro. The final goal is to determine a restriction nuclease map of the chloroplast DNA, and to undertake physical mapping of chloroplast genes by in situ hybridization techniques.